一种重组蛋白A的定点固定化方法
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- 英文论文题名：Direct site-specific immobilization of protein A via aldehyde-hydrazide conjugation
- 论文作者：臧柏林 ; 任军 ; 徐丽 ; 贾凌云
- 英文论文作者：Belin Zang ; J Ren ; L Xu ; L Jia ; School of life science and biotechnology ; Dalian University of Technology
- 年：2016
- 作者机构：大连理工大学生命科学与技术学院;
- 论文关键词：定点固定化 ; 蛋白A色谱 ; 甲酰甘氨酸生成酶 ; 位点特异性修饰
- 会议召开时间：2016-07-01
- 会议录名称：中国化学会第30届学术年会摘要集-第二十三分会：复杂样品分离分析
- 语种：中文
- 分类号：O629.73
- 学会代码：ZGHY
- 会议名称：中国化学会第30届学术年会-第二十三分会 ; 复杂样品分离分析
- 会议地点：中国辽宁大连
- 主办单位：中国化学会
- 学会名称：中国化学会
- 页数：1
- 文件大小：125k
- 原文格式：D
- 会议级别：全国
摘要
蛋白质固定化是亲和色谱,固定化酶等应用的关键技术之一。有效的固载方法能够在提高载量的同时最大程度的保持蛋白质配基的活性。现有的固载方法多采用蛋白表面天然氨基酸侧链基团与固相基质表面的活性基团进行化学反应偶联。由于蛋白表面通常含有多个可供偶联的侧链基团,所以化学偶联法不具有选择性,易使蛋白活性损失。本文介绍了一种简单的通过蛋白定点醛基化修饰从而定点固载的方法。该方法采用蛋白A为模式蛋白,在其N端加入甲酰甘氨酸生成酶(FGE)识别序列,与FGE在大肠杆菌内共表达,实现FGE催化体内蛋白A定点醛基化修饰。由于一般的蛋白不含有醛基,所以能利用酰肼与醛基在温和条件下的特异性反应直接从细胞破碎上清中固载蛋白A。这种方法省去了蛋白配基的纯化过程,降低了成本;同时又实现了定点固定化,最大程度保留蛋白的活性,具有广阔的工业化应用前景。
Immobilization of protein ligands on supporting matrices is a key step for the preparation of affinity chromatography resins,immobilized enzyme-reactors and other application.An efficient coupling strategy can significantly improve the validity and cost of the affinity system,especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands.This study described a simple method for obtaining site-specific immobilization of protein A(the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography.An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme(FGE) and recombinant protein A containing a FGE recognizing sequence(aldehyde tag) in Escherichia coli.The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands,but also improved the cost of the process by employing unpurified ligands,a method that might be of great use to industrial applications.
Immobilization of protein ligands on supporting matrices is a key step for the preparation of affinity chromatography resins,immobilized enzyme-reactors and other application.An efficient coupling strategy can significantly improve the validity and cost of the affinity system,especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands.This study described a simple method for obtaining site-specific immobilization of protein A(the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography.An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme(FGE) and recombinant protein A containing a FGE recognizing sequence(aldehyde tag) in Escherichia coli.The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands,but also improved the cost of the process by employing unpurified ligands,a method that might be of great use to industrial applications.
引文
[1]Zang,B.;Ren,J;Xu,L;Jia,L*.J Chromatogr B,2016,1008:132.